The long-term goal of this program is to elucidate those molecular mechanisms of mammalian lens development and homeostasis that are directly controlled by DNA-binding transcription factor Pax6. Previous studies have shown that Pax6 is essential for establishing lens lineage and regulation of crystallin gene expression. However, the complete spectrum and range of Pax6's function and the mechanism by which it affects lens development remain to be determined. Using chromatin immunoprecipitation (ChIP), we have now identified a large number of novel genes including Mab21l1 and DNase II2 as putative direct Pax6 targets. Mab21l1 is highly expressed in the lens placode and its promoter region contains multiple binding sites for Six3, another important lens-lineage specific regulatory gene. DNase II2 is most highly expresed in lens fiber cells just prior to their denucleation. Evidence exists that lens-preferred expression of this gene is under the control of Pax6, AP-21 and Foxe3. In contrast, Hsf4 plays a direct role in the upregulation of DNase II2 in differentiating lens fiber cells. We also found that Pax6 not only promotes lens development but it also simultanously suppresses alternative developmental programs such as the expression of neurogenic genes in lens lineage. These findings suggest that Pax6 controls epigenetic mechanims that control individual cell lineage formation in embryonic development. In order to carry out this long-term goal, the following specific aims are proposed: (1) To elucidate transcriptional regulation of Mab21l1, a gene essential for the survival of lens progenitor cells, by Pax6, Six3 and other factors in transgenic mouse and via cell culture experiments. (2) To elucidate transcriptional regulation of DNase II2, an enzyme required for lens fiber cell denuclation, by Pax6, AP-21, Foxe3 and Hsf4, in transgenic mouse and through a combination of protein-DNA binding studies and cell culture based reporter assays. (3) To identify those novel direct Pax6-targets that are regulated via distal 5'and 3'enhancers and to generate a Pax6-dependent regulatory network that controls lens and forebrain development using chromatin immunoprecipitations analyzed by massively parallel DNA sequencing (ChIP-seq), RNA expression profiling in normal and Pax6 mutated tissues. PUBLIC HEALTH RELEVANCE: This application is relevant to human health as lens cataract is a major cause of worldwide blindness. Pax6 is an essential gene for mammalian eye development, it controls lens lineage formation and terminal differentiation of lens fiber cells. Mutations in PAX6 and other genes studied here (FOXE3, HSF4 and MAF) are known to cause human congenital eye diseases. Mutations in PAX6 also cause a variety of neurological disorders and PAX6 has also been implicated in type II diabetes.